1. Field of the Invention
This invention is related to the amplitude and quantitative phase imaging using digital holography of digital holographic microscopy for real time 3D measurements.
2. Description of Related Art
Digital holography is an effective tool to record not only the amplitude image but also the phase image contrary to the normal imaging techniques. The main advantage of digital holography is obviously originated from the real time 3D measurements of the target with only a single image. In addition, digital holography allows reconstruction of the amplitude and phase of the object wave even though the specimen is not in the best focus or the optical system includes aberrations.
Based on these advantages, recently, digital holography has been widely used in industrial and science fields, especially in biomedical fields where the amplitude and phase images of live cells should be monitored in real time. In manufacturing industrial products, digital holography can raise the throughput significantly with the real time inspection of the products topographically as well as tomographically.
The principle of digital holography starts from the traditional holography, based on two-step process, recording and reconstruction. In the recording process, the scattered wave from the object interferes with the reference wave and the interference pattern is recorded in a photosensitive media, i.e. it generates ‘hologram’. After the recording process, reconstruction of the object image is carried out by illuminating the same reference wave to the hologram without the object.
Similar to this, digital holography also has the same procedures as traditional holography, but it uses a video camera or optical sensor as a recording media. Moreover, the digital hologram allows a numerical image reconstruction wherein the physical reconstruction wave is simulated virtually and the object wave can be recovered by numerical wave propagations.
Digital holography (DH) can be typically categorized in off-axis DH and in-line DH, according to the optical configuration, i.e. the alignment between reference and measurement waves. In order to avoid a DC term (zero-order diffraction) and twin image term (conjugate of the object wave) overlapping the object wave term, off-axis DH uses a spatial modulation and spatial filtering technique with a tilt angle between reference and measurement waves. Without the effort that in-line DH takes to eliminate these noise effects, off-axis DH allows reconstruction of the amplitude and phase image of the specimen from the hologram.
On the other hand, in-line DH typically adopts other techniques such as phase shifting to extract only the object wave from the hologram. Inherently, in-line DH loses the main strength such as real time measurement ability in the applications, although in-line DH allows clear reconstructing of the image. Recently, in order to overcome this limitation, parallel optical-path-length-shifting DH based on the spatial phase shifting device has been proposed for real time measurements. However, this method is limited by the available number of pixels in the CCD camera or optical sensor.
In the off-axis DH, most widely used in the industrial and science fields, the spatial modulation of the image caused by the tilt of the reference mirror is introduced in the digital hologram as shown in FIG. 1 and it is used for the filtering process to extract the object wave in the spatial frequency domain. The configuration depicted in FIG. 1 comprises a light source 1, like a laser light source, a beam splitter 3, a condensing lens 2 located between the light source 1 and the beam splitter 3, a reference mirror 5, a first objective lens 4 located an imaging lens 6, an optical sensor 7, a second objective lens 8 and holder for a sample 9.
The light source 1 emits a first reference beam 10 passing the condensing lens 2, the beam splitter 3, the first objective lens 4, the reference mirror 5, where it is reflected, again the first objective lens 4, the beam splitter 3 where the reference beam is deflected to the imaging lens 6 and it hits the optical sensor 7.
Further the light source 1 emits a measurement light beam 11 which passes the condensing lens 2, is deflected by the beam splitter 3, passes the second objective lens 8, hits the sample 9, is reflected by sample 9, passes again the second objective lens 8, the beam splitter 3, the imaging lens 6 and finally hits the sensor 7 to form an interference pattern together with the first reference beam 10.
The main advantage of the off-axis configuration is the ability to obtain the objective wave with a single hologram contrary to the other configurations such as inline digital holography and phase shifting digital holography. After obtaining a digital hologram which contains interference fringes by the off-axis geometry, the two-dimensional spectrum of the hologram can be obtained by the Fourier transform in the spatial frequency domain, where the different terms of the interference produce well-separated contributions. The spectrum of a DC term, which is a depicted as the spectrum in the centre of FIG. 5A, represents the spectrum of the intensities for reference and object waves (no interference terms).
The spatial frequencies of the interference terms are located symmetrically with respect to the centre of the image. Their distances to the centre depend on the incidence tilt angle, which must be large enough to ensure a complete separation of the DC term from those of the interference terms. Then the unwanted terms can be filtered out in the spatial frequency domain and only the object wave can be extracted and obtained although the spatial frequency is limited in spite of using a high resolution camera. After filtering, the amplitude and phase image of the object are obtained by numerical reconstruction of the hologram with a plane wave as a reference wave.
More in particular the prior art discloses a method for preparing a digital hologram representing an image of an object, the method comprising the steps of generating a coherent measurement beam and a first coherent reference beam by a light source, irradiating the object by the measurement beam and guiding the measurement beam reflected by the object to an optical sensor, guiding the first reference beam to a first mirror extending under an angle different from 90° with the optical axis of the first reference beam and guiding the first reference beam reflected by the first mirror to the optical sensor so that the measurement beam and the first reference beam together generate an interference pattern on the sensor, reading out the optical sensor and providing a digital signal representing the interference pattern generated on the optical sensor, processing the digital signal to obtain a digital hologram, subjecting the digital hologram to a Fourier transform in the spatial frequency domain to obtain a two dimensional spectrum comprising a DC-term, a first image term and a first conjugate image term, and subjecting the resulting spectrum to filtering to obtain a term representing the object.
The most important procedure in the off-axis DH is the spatial filtering process to eliminate other terms, i.e. a DC term (zero-order) and a twin image term (conjugate wave), and to obtain the high quality object wave from the digital hologram.
T. M. Kreis describes a simple method to suppress the DC term from the hologram (T. M. Kreis and W. P. P. Jüptner, “Suppression of the dc term in digital holography,” Opt. Eng. 36, 2357-2360, 1997). This method consists in subtracting the mean intensity from the digital hologram, which permits only the elimination of the so-called DC term from the reconstructed images. It's a simple way to reduce the DC terms from the hologram but it's not sufficient in most of cases. If the object wave intensity is not constant in hologram plane in general, for example, the DC term caused by the object wave cannot be eliminated with this method.
E. Cuche proposed an improved approach known as spatial filtering used in the form of a band-pass filter (E. Cuche et al., “Spatial filtering for zero-order and twin-image elimination in digital off-axis holography,” Appl. Opt. 39 (23), 4070-4075, 2000).
U.S. Pat. No. 6,262,818 to Cuche et al introduces the spatial filtering methods in two ways; one as the band-pass filtering method in the spatial frequency domain using FFT and the other as the optical spatial filtering method based on 4-f system. These methods depend on the critical assumption that the DC term and the desired term are well separated so that the DC term can be suppressed by filtering. However, they are also limited by these two aspects; one is that a certain fraction of the spectrum can be available and the other is that the spatial filtering often requires manual intervention for selecting the desired order.
Another method to achieve the effective filtering using additional images is disclosed in U.S. Pat. No. 6,809,845 to Kim et al. In this method, the reference wave intensity and object wave intensity are obtained in addition to the hologram in the system and these are used for removing the DC terms of the hologram by simple subtraction.
However, it needs additional hardware such as beam blockers and should record two more images except the hologram. In this case, it is assumed that the environmental conditions and system parameters should be kept constantly.
Recently, a nonlinear reconstruction technique has been introduced (N. Pavillon et al., “Suppression of the zero-order term in off-axis digital holography through nonlinear filtering,” Appl. Opt. 48 (34), H186-H195, 2009). It enables exact zero-order free reconstruction in off-axis DHM even if the zero-order and the object wave spectra overlap. The nonlinear filtering technique works under two realistic assumptions on the digital hologram; first, the spectrum of the object wave should be confined to a quadrant of the Fourier domain and second, the intensity of the object wave should be much smaller than that of the reference.
However, the small intensity of the object wave can lower the visibility of the interference fringe and even a signal to noise ratio (SNR), which can cause other errors. It means the effectiveness of this method can be limited in the practical applications.
On the other hand, the suppression of the zero-order term by employing the information obtained during wavefront reconstruction in an iterative procedure was disclosed (N. Pavillon et al., “Iterative method for zero-order suppression in off-axis digital holography,” Opt. Express 18 (15), 15318-15331, 2010). Consequently, it enables the DC term suppression without any a priori knowledge about the object.
However, this technique takes the calculation time until reaching to an acceptable error level as its definition.